造血干细胞多药耐药P-糖蛋白的表达及多药耐药逆转剂对其功能的影响

了解多药耐药Ｐ－糖蛋白在造血干／祖细胞的表达,探讨多药耐药逆转剂在临床应用过程中可能对造血干细胞的损害。方法：采用ＭＲＫ１６单克隆抗体,利用流式细胞仪测定健康人骨髓ＣＤ３４阳性细胞表达Ｐ－ｇｐ的表达;将多药耐药逆转剂ＭＳ－２０９作用于造血细胞,检测ＭＳ－２０９对ＣＤ３４阳性细胞摄取染料Ｒｈｏｄａｍｉｎ－１２３的影响。     

逆转MDR-1基因相关的消化系统肿瘤多药耐药

消化系统恶性肿瘤细胞多药耐药是导致化疗失败的重要原因.多药耐药基因(MDR-1)过度表达P-糖蛋白(p-gp)是产生耐药的主要机制.逆转MDR-1基因相关的消化系统肿瘤多药耐药,提高肿瘤细胞对化疗药物的敏感性的方式有两类一是直接抑制p-gp的药泵功能;二是干扰MDR-1基因的表达.     

环氧合酶-2、P-糖蛋白与肿瘤多药耐药

P 糖蛋白介导的肿瘤多药耐药是肿瘤细胞产生耐药性主要的机制之一。环氧合酶 2 是肿瘤发生的限制点之一,参与肿瘤发生发展的多个环节。近年来一些研究表明环氧合酶 2 系统可能参与 P 糖蛋白的表达。该文对环氧合酶 2与 P 糖蛋白的关系作一综述。     

环氧合酶-2、P-糖蛋白与肿瘤多药耐药

P-糖蛋白介导的肿瘤多药耐药是肿瘤细胞产生耐药性主要的机制之一。环氧舍酶-2是肿瘤发生的限制点之一，参与肿瘤发生发展的多个环节。近年来一些研究表明环氧舍酶-2系统可能参与P-糖蛋白的表达。该文对环氧舍酶-2与P-糖蛋白的关系作一综述。     

肺癌多药耐药现象与生物膜表面糖蛋白

肺癌化疗前后常产生多药耐药使化疗失败,目前认为生物膜上某些糖蛋白与多药耐药有关,本文拟对这些糖蛋白的结构、功能及与肺癌的多药耐药机制加以概述。     

槲皮素逆转肿瘤多药耐药作用研究进展

槲皮素是一种具有多种生理活性和药理活性的天然小分子黄酮类化合物。近年来研究发现,槲皮素可抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡、干扰肿瘤细胞周期、干扰肿瘤细胞信号转导通路、逆转肿瘤细胞多药耐药等作用。本文结合国内外文献,就槲皮素影响肿瘤细胞多药耐药相关蛋白的表达,逆转肿瘤多药耐药方面进行综述分析。     

中药单体逆转肿瘤多药耐药的研究进展

肿瘤多药耐药(multidrug resistance,MDR)是目前肿瘤临床化疗失败的主要原因之一,中药因其低毒高效和多阶段性作用的优势已在肿瘤MDR逆转方法的研究中逐步受到重视.目前已从多种中药中发现了具有逆转肿瘤MDR作用的中药单体成分.本文对肿瘤MDR的形成机制及近年来中药单体在逆转肿瘤MDR领域的最新研究成果进行综述.     

环孢素A逆转肿瘤多药耐药的临床观察

多药耐药（MDR）是恶性肿瘤化疗失败的原因之一,临床许多病人在经历了最初有效的化疗之后,最终仍难免于复发,此时再使用曾有效的药物或从未用过的药物,均不见疗效.其主要原因是肿瘤细胞对化疗产生了耐药性,多药耐药基因（MDR1）呈高表达.因此,逆转肿瘤细胞的耐受性是提高肿瘤化疗疗效的关键.国内外许多基础研究表明,很多药物可用来逆转肿瘤细胞的MDR.我们自2002年起用RT-PCR法检测MDR1表达,用免疫调节剂环孢素A口服逆转肿瘤MDR取得良好效果.     

肝癌耐药细胞中MDR1 mRNA、P-糖蛋白的表达变化及意义

目的观察肝癌耐药细胞中MDR1 mRNA及P-糖蛋白的表达变化,并探讨其临床意义。方法采用药物浓度递增法,诱导产生对盐酸阿霉素（Adr）具有稳定耐药性的肝癌HepG2细胞（HepG2Adr）;采用RT—PCR及免疫组化SP法,分别检测HepG2、HepG2Adr细胞中的MDR1 mRNA、P-糖蛋。结果经0．01、0．10、1．00、10．00μmol／LAdr诱导产生的HepG2Adr细胞中MDR1 mRNA的表达量分别为0．26±0．11、0．56±0．17、10．78±0．2、1．21±0．17,P-糖蛋白阳性表达率分别为15．44％±4．55％、26．76％±5．23％、64．21％±14．22％、90．23％±7．68％;HepG2细胞中MDR1 mRNA、P-糖蛋白分别为0．18±0．08、5．73％±0．52％。肝癌HepG2Adr细胞与HepG2细胞中MDR1 mRNA、P-糖蛋白表达量比较,P均〈0．05;且随着肝癌HepG2Adr细胞耐药性增高,MDR1 mRNA、P-糖蛋白表达量逐渐增高（P均〈0．05）。结论肝癌耐药细胞中MDR1 mRNA及P-糖蛋白高表达,二者可能与肝癌多药耐药的产生有关。     

膜糖蛋白介导的肺癌耐药与逆转机制的研究现状

尽管近年来肺癌的诊疗技术有了很大提高，但5年生存率仍不足15％，其主要原因是化疗对肺癌，尤其是非小细胞肺癌（non-small-cell lung cancer，NSCLC）效果不佳，多药耐药（muhidrug resistance，MDR）是其主要原因。MDR是指肿瘤细胞对一种化疗药物产生耐药现象后，对其他结构、细胞靶点和作用机制迥然不同的化疗药物产生交叉耐药。肺癌的耐药机制比较复杂，     

Intercellular transfer of P-glycoprotein mediates acquired multidrug resistance in tumor cells

The overexpression of P-glycoprotein (P-gp) causes resistance to chemotherapy in many tumor types. Here, we report intercellular transfer of functional P-gp from P-gp-positive to P-gp-negative cells in vitro and in vivo. The expression of acquired P-gp is transient in isolated cells but persists in the presence of P-gp-positive cells or under the selective pressure of colchicine. The intercellular transfer of functional P-gp occurs between different tumor cell types and results in increased drug resistance both in vitro and in vivo. Most importantly, the acquired resistance permits tumor cells to survive potentially toxic drug concentrations long enough to develop intrinsic P-gp-mediated resistance. P-gp transfer also occurs to putative components of tumor stroma, such as fibroblasts, raising the possibility that multidrug resistance could be conferred by resistant tumor cells to critical stromal elements within the tumor mass. This is the first report, to our knowledge, that a protein transferred between cells retains its function and confers a complex biologic property upon the recipient cell. These findings have important implications for proteomic analyses in tumor samples and resistance to cancer therapy.     

Imaging reversal of multidrug resistance in living mice with bioluminescence: MDR1 P-glycoprotein transports coelenterazine

Coelenterazine is widely distributed among marine organisms, producing bioluminescence by calcium-insensitive oxidation mediated by Renilla luciferase (Rluc) and calcium-dependent oxidation mediated by the photoprotein aequorin. Despite its abundance in nature and wide use of both proteins as reporters of gene expression and signal transduction, little is known about mechanisms of coelenterazine transport and cell permeation. Interestingly, coelenterazine analogues share structural and physiochemical properties of compounds transported by the multidrug resistance MDR1 P-glycoprotein (Pgp). Herein, we report that living cells stably transfected with a codon-humanized Rluc show coelenterazine-mediated bioluminescence in a highly MDR1 Pgp-modulated manner. In Pgp-expressing Rluc cells, low baseline bioluminescence could be fully enhanced (reversed) to non-Pgp matched control levels with potent and selective Pgp inhibitors. Therefore, using coelenterazine and noninvasive bioluminescence imaging in vivo, we could directly monitor tumor-specific Pgp transport inhibition in living mice. While enabling molecular imaging and high-throughput screening of drug resistance pathways, these data also raise concern for the indiscriminate use of Rluc and aequorin as reporters in intact cells or transgenic animals, wherein Pgp-mediated alterations in coelenterazine permeability may impact results.     

表没食子儿茶素没食子酸酯逆转人白血病细胞多药耐药性及机制研究

目的：探讨表没食子儿茶素没食子酸酯[(-)epigallocatechin-3-gallate(EGCG）对人自血病细胞K562／A02多药耐药性的逆转作用及相关机制。方法：采用MTT法确定EGCG的非细胞毒性剂量。以及对K562／A02细胞药物敏感性的影响。以流式细胞仪测定细胞内化疗药物浓度的改变，同时于逆转前后采用免疫细胞化学方法检测凋亡相关蛋白bcl-2和bax表达水平的变化。结果：EGCG在低于103．2μmol/L时对K562/A02细胞的生长抑制率小于10％。40μmol/L、60μmol/L及80μmol/L EGCG均可增加K562／A02细胞对阿霉素的敏感性，使阿霉素对K562／A02细胞的IC50由原来的113．34mg／L降低至9．66mg／L、7．67mg/L和4．68mg／L，其逆转倍数分别为11．73、14．77和24．23倍。同时，不同浓度EGCG均可增加细胞内阿霉素浓度，并呈剂量依赖性。在40μmol/L、60μmol/L及80μmol/L EGCG作用后，细胞bcl-2和bax的表达水平均增加，并且bax/bcl-2比值升高。结论：EGCG可部分逆转人白血病细胞K562／A02对阿霉素的耐药性，其逆转机制与增加细胞内化疗药物浓度，升高细胞bax／bcl-2比值从而诱导细胞凋亡有关。     

Reversing the direction of drug transport mediated by the human multidrug transporter P-glycoprotein

P-glycoprotein (P-gp), also known as ABCB1, is a cell membrane transporter that mediates the efflux of chemically dissimilar amphipathic drugs and confers resistance to chemotherapy in most cancers. Homologous transmembrane helices (TMHs) 6 and 12 of human P-gp connect the transmembrane domains with its nucleotide-binding domains, and several residues in these TMHs contribute to the drug-binding pocket. To investigate the role of these helices in the transport function of P-gp, we substituted a group of 14 conserved residues (seven in both TMHs 6 and 12) with alanine and generated a mutant termed 14A. Although the 14A mutant lost the ability to pump most of the substrates tested out of cancer cells, surprisingly, it acquired a new function. It was able to import four substrates, including rhodamine 123 (Rh123) and the taxol derivative flutax-1. Similar to the efflux function of wild-type P-gp, we found that uptake by the 14A mutant is ATP hydrolysis-, substrate concentration-, and time-dependent. Consistent with the uptake function, the mutant P-gp also hypersensitizes HeLa cells to Rh123 by 2- to 2.5-fold. Further mutagenesis identified residues from both TMHs 6 and 12 that synergistically form a switch in the central region of the two helices that governs whether a given substrate is pumped out of or into the cell. Transforming P-gp or an ABC drug exporter from an efflux transporter into a drug uptake pump would constitute a paradigm shift in efforts to overcome cancer drug resistance.

The emergence of drug resistance in cancer is a major clinical obstacle. The multidrug-resistance–linked plasma membrane transporter ABCB1 or P-glycoprotein (P-gp) effluxes various toxic amphipathic and hydrophobic compounds from cells, including anticancer drugs (1–4). Structurally, P-gp is comprised of two transmembrane domains (TMDs), each having six transmembrane helices (TMHs) and one cytosolic nucleotide-binding domain (NBD). Recent cryo-electron microscopy (cryo-EM) studies have shown that human P-gp exists in both inward open and inward closed conformations (5, 6). Substrates bind to P-gp in the inward open conformation and are then effluxed. The process is associated with conformational changes induced by ATP binding and hydrolysis by the NBDs (7).Earlier studies revealed that the transport of substrates mediated by P-gp involves a number of specific residues in the transmembrane (TM) region. Some of these residues directly interact with the substrates, while others help to achieve the proper conformation of the protein for the translocation process. Previously, we generated P-gp mutants with single, double, or triple mutations that alter the binding, but not the transport of various substrates (8). We also generated a P-gp variant with 15 residues in the drug-binding pocket mutated to tyrosine that could transport most of the substrates despite increased hydrogen-bonding potential (9). These studies revealed the high degree of permissiveness of the drug-binding pocket of P-gp. To further investigate the mechanism of this transporter we decided to introduce multiple mutations in a pair of homologous helices known to be involved in the binding and translocation of substrates. Using this strategy, we generated a mutant named TMH 1,7 that has six mutations each in TMH 1 and TMH 7. This mutant has diminished polyspecificity, as it is able to transport only 3 of the 25 substrates tested (10).In this study, we focus on another pair of homologous helices, TMH 6 and TMH 12, which form part of the drug-binding cavity through which substrates are translocated. To test the hypothesis that mutations in multiple residues in these homologous TMHs would significantly modify the drug-binding pocket resulting in altered or loss of binding and transport of substrates, the 14A mutant of human P-gp was generated in which seven conserved residues from TMH 6 and seven conserved residues from TMH 12 were mutated to alanine. Our results showed that the 14A mutant could not efflux most of the substrates tested. While P-gp is known to be able to pump many diverse toxic substances out of cells, we were surprised to find that the 14A mutant actually gained the ability to import certain substrates including rhodamine123 and flutax-1. So in effect, the mutations had caused a change in the direction of transport for those substrates. We found that this uptake function of the 14A mutant is ATP-binding– and hydrolysis-dependent, and similar to other ABC importers, the mutant transporter exhibits a narrow substrate specificity. Further mutagenesis of additional residues identified a possible switch in the central region of these two helices that determines the direction of the substrate transport. Our findings suggest that the ability to change the direction of transport of an ABC drug transporter from efflux to influx might provide a strategy to kill cancer cells specifically expressing such transporters.     

Expression and activity of the multidrug resistance P-glycoprotein in human peripheral blood lymphocytes.

P-glycoprotein (P-gp), the product of the MDR1 (multidrug resistance) gene, is a transmembrane efflux pump for different lipophilic compounds, including many anticancer drugs and fluorescent dyes. We have previously reported that the efflux of fluorescent dyes from lymphoid cells of human bone marrow was directly correlated with the cellular P-gp content. In the present study, we show that human peripheral blood lymphocytes (PBL) also express P-gp, and that P-gp expression correlates with the efflux of fluorescent dyes from PBL. This efflux was suppressed not only by chemical inhibitors of P-gp but also by a P-gp-specific monoclonal antibody UIC2, thus providing direct evidence that it was mediated by P-gp. We have also characterized dye efflux and UIC2 reactivity in specific PBL subsets. P-gp was expressed in the majority of CD56+, CD8+, and CD20+ lymphocytes, but in less than one half of CD4+ cells. P-gp-mediated dye efflux was highly heterogeneous relative to the expression of CD56RA, CD56RO, Leu-8, and HLA-DR antigens. No significant P-gp activity was detectable in CD14+ monocytes. MDR1 expression in normal lymphocytes may be a determinant of multidrug resistance in the corresponding malignancies.     

Amino acid substitutions in the sixth transmembrane domain of P-glycoprotein alter multidrug resistance.

Eukaryotic cells can display resistance to a wide range of natural-product chemotheraputic agents by the expression of P-glycoprotein (pgp), a putative plasma membrane transporter that is thought to mediate the efflux of these agents from cells. We have identified, in cells selected for multidrug resistance with actinomycin D, a mutant form of pgp that contains two amino acid substitutions within the putative sixth transmembrane domain. In transfection experiments, this altered pgp confers a cross-resistance phenotype that is altered significantly from that conferred by the normal protein, displaying maximal resistance to actinomycin D. These results strongly implicate the sixth transmembrane domain in the mechanism of pgp drug recognition and efflux. Moreover, they indicate a close functional homology between pgp and the cystic fibrosis transmembrane regulator in which the sixth transmembrane domain has also been shown to influence substrate specificity.     

结核病的流行特征与耐多药的窘迫及其策略

<正>结核病(tuberculosis,TB)是由结核分枝杆菌(Mycobacterium tuberculosis,MTB)感染引起的全球性慢性呼吸道传染病[1-3]。结核病是伴随人类历史最长,数千年来夺走了人类不计其数的鲜活生命[4]。结核病是我国重点控制的重大疾病之一,也是全球关注的公共卫生和社会问题。尽管我国结核病防治工作取得了很大成效,但结核病的流行仍然较为严重,结核病是当前威胁人类健康的重大传染性疾病[5-6]。虽然20世纪抗结药物     

Cell biological mechanisms of multidrug resistance in tumors.

Multidrug resistance (MDR) is a generic term for the variety of strategies tumor cells use to evade the cytotoxic effects of anticancer drugs. MDR is characterized by a decreased sensitivity of tumor cells not only to the drug employed for chemotherapy but also to a broad spectrum of drugs with neither obvious structural homology nor common targets. This pleiotropic resistance is one of the major obstacles to the successful treatment of tumors. MDR may result from structural or functional changes at the plasma membrane or within the cytoplasm, cellular compartments, or nucleus. Molecular mechanisms of MDR are discussed in terms of modifications in detoxification and DNA repair pathways, changes in cellular sites of drug sequestration, decreases in drug-target affinity, synthesis of specific drug inhibitors within cells, altered or inappropriate targeting of proteins, and accelerated removal or secretion of drugs.     

Immunosuppressants FK506 and rapamycin function as reversal agents of the multidrug resistance phenotype.

The multidrug-resistant (MDR) phenotype is characterized in vitro by the resistance displayed by cell lines to a broad spectrum of natural product cytotoxic agents. This high level of cross-resistance is due to the increased expression of a membrane glycoprotein termed P-glycoprotein. Encoded in humans by the mdr1 gene, P-glycoprotein functions as an energy-dependent efflux pump of these cytotoxic agents. In this report, we demonstrate that the newly characterized immunosuppressant FK506 and its structural analogue, rapamycin, are capable of functioning as MDR reversal agents. FK506 and rapamycin increase both intracellular, cytotoxic drug (daunomycin) accumulation, and the cytotoxicity of chemotherapeutic agents in multidrug-resistant cells. The increase in cytotoxic drug accumulation is observed at concentrations of FK506 and rapamycin 1,000-fold greater than the concentrations required for FK506 and rapamycin to inhibit T-lymphocyte activation and similar to those shown to be effective for other MDR reversal agents such as cyclosporine A (CsA) and verapamil. The effect of FK506 or rapamycin on both intracellular accumulation and cytotoxicity of daunomycin is additive. This is supported by the ability of FK506 and rapamycin to directly compete the binding of the photoaffinity analogue 125I-iodoaryl azidoprazosin to the P-glycoprotein. The data demonstrate that FK506 and rapamycin represent a new class of structurally distinct molecules that can function as MDR reversal agents and suggest a previously unidentified, potential clinical role for these compounds.